In vitro analysis of exfoliated cells plays an important role in disease screening and clinical diagnosis. An accurate cytological diagnosis depends largely on the ability to detect changes in cellular and nuclear structural characteristics of individual single cells. The diffraction-limited resolution of a conventional light microscope limis the detection of mostly micron-scale features of cancer cells, which result in intra-observer variation, missed early-stage cancers (false negatives), and indeterminate cases that often result in unnecessary invasive procedures in the absence of cancer (false positives). Technologies that perform high-speed, accurate, quantitative and cost-effective cytological analysis and are suitable for routine clinic use are urgently needed. Cellular nanoscale structural changes represent a promising strategy for identifying pre-cancerous or cancerous cells. We have recently developed two complementary techniques, both of which can assess nanoscale structural characteristics at the single-cell level: quantitative phase microscopy and spectral-encoding of spatial frequency (SESF). We demonstrated their ability to detect structural changes in pre-cancerous cells missed by conventional light microscopy. We propose to develop a new advanced microscopy that integrates the complementary attributes of these two techniques to perform real-time quantitative structural imaging and high-speed and comprehensive nanoscale structural analysis of label-free single cells in their natural state for use in both research and clinical applications. The technical performance of this microscopy system will be characterized and their ability to analyze nanoscale structures will be rigorously validated. It will be optimized for routine clinical use to analyze unstained exfoliative cytology specimens by developing cell segmentation algorithm and multi-metric structural analysis. We will focus on two large-volume and clinically challenging cytology specimens as our disease models: cervical and urine cytology. The accuracy of the system will be tested using a relatively large scale of cervical and urine cytology specimens via a training and a validation set. If successful, this technique could be used to support conventional cytology for accurate, quantitative, high-speed, comprehensive and cost-effective cytological analysis to improve the diagnostic accuracy and prevent costly unnecessary procedures.